A kind of steviol derivative and its preparation method and application

Abstract

The invention discloses a steviol derivative, a preparation method thereof and an application thereof in preparing a cardioprotective drug; the invention aims to provide a steviol derivative having cardioprotective activity; the C-19 position of steviol is modified, a series of substituted benzyl ester fragments are introduced, and a new steviol derivative is obtained, which shows good cardioprotective activity in a zebrafish model of myocardial injury and can be used for preparing a cardioprotective drug; the invention relates to the fields of medical technology and pharmaceutical chemistry.

Description

Technical Field

[0001] The present invention relates to the field of drug design and medicinal chemistry, and specifically to a steviol derivative. The present invention also relates to a preparation method and application of the steviol derivative. Background Art

[0002] Cardiovascular diseases include coronary heart disease, rheumatic heart disease, myocardial infarction, cardiomyopathy, congenital heart disease and thromboembolic disease.

[0003] Drug development based on natural products is an important branch of drug research and development. Compared with synthetic compounds, natural products show more complex and rich diversity in chemical structure. Because they are produced in nature, their chemical structures and functions have been selected and optimized in the long-term evolution process. These unique chemical structures give many natural products the ability to specifically bind to specific targets and show good biological activity, allowing them to be directly used as drugs for the treatment of diseases.

[0004] Steviol is an enantiokaurane-type diterpene and is the alkaline hydrolysis product of the sweetener steviolside. Steviol has a wide range of biological activities, including anti-tumor, myocardial protection, antibacterial, and anti-inflammatory effects. Currently, the structural modification of steviol's cardioprotective activity is still very limited. Summary of the invention

[0005] One of the objects of the present invention is to provide a steviol derivative having cardioprotective activity.

[0006] A second object of the present invention is to provide a method for preparing steviol derivatives.

[0007] The third object of the present invention is to provide the use of steviol derivatives in the preparation of cardioprotective drugs.

[0008] To this end, the first technical solution provided by the present invention is a steviol derivative having a general structural formula 1:

[0009]

[0010] Wherein: R is one of m-fluorobenzyl, 4-trifluoromethylbenzyl, 2,3-difluorobenzyl, 3,5-difluorobenzyl, o-fluorobenzyl, p-chlorobenzyl, m-chlorobenzyl, 2,4-difluorobenzyl, m-iodobenzyl, p-fluorobenzyl, 3-methylbenzyl, 3-nitrobenzyl, m-bromobenzyl, 3-phenylbenzyl, 3-trifluoromethylbenzyl, 2,3,4,5,6-pentafluorobenzyl, 3,4-difluorobenzyl, 3,4-dimethoxybenzyl, 3-methoxybenzyl, 2,5-difluorobenzyl, 3-difluoromethoxybenzyl, o-chlorobenzyl, 3-cyanobenzyl, 3,4-dichlorobenzyl, 3-trifluoromethoxybenzyl, and 3-aldehydebenzyl.

[0011] To this end, the first technical solution provided by the present invention is as follows:

[0012] A steviol derivative having a general structural formula of Formula 1:

[0013]

[0014] Wherein: R is one of m-fluorobenzyl, 4-trifluoromethylbenzyl, 2,3-difluorobenzyl, 3,5-difluorobenzyl, o-fluorobenzyl, p-chlorobenzyl, m-chlorobenzyl, 2,4-difluorobenzyl, m-iodobenzyl, p-fluorobenzyl, 3-methylbenzyl, 3-nitrobenzyl, m-bromobenzyl, 3-phenylbenzyl, 3-trifluoromethylbenzyl, 2,3,4,5,6-pentafluorobenzyl, 3,4-difluorobenzyl, 3,4-dimethoxybenzyl, 3-methoxybenzyl, 2,5-difluorobenzyl, 3-difluoromethoxybenzyl, o-chlorobenzyl, 3-cyanobenzyl, 3,4-dichlorobenzyl, 3-trifluoromethoxybenzyl, and 3-aldehydebenzyl.

[0015] The second technical solution provided by the present invention is a method for preparing the steviol derivative in Formula 1, which comprises the following steps in sequence:

[0016] (1) Synthesis of compounds 2a-z

[0017] 0.47 mmol of compound 1, 0.7 mmol of potassium carbonate and 0.7 mmol of halogenated compound were dissolved in 3 mmol of acetone, and the mixture was stirred under reflux at 60°C for 5 h; a mixture of ethyl acetate and saturated brine was added for extraction, and the organic layer was collected, dried over anhydrous sodium sulfate, concentrated, and separated and purified by column chromatography to obtain compounds 2a-z;

[0018] Its synthetic route is as follows:

[0019]

[0020] Wherein R in compound 2a-z is one of the following substituents:

[0021]

[0022] The last technical solution provided by the present invention is the use of the above-mentioned steviol derivatives in the preparation of cardioprotective active drugs.

[0023] The present invention also provides a cardioprotective active drug, comprising a steviol derivative having a general structural formula of Formula 1.

[0024] More preferably, the drug further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH adjuster.

[0025] The dosage form of the medicine is injection, tablet, oral solution, granule or capsule.

[0026] Compared with the prior art, the present invention modifies the 19th position of the natural product steviol and introduces a series of substituted benzyl ester fragments to obtain a new steviol derivative with significant cardioprotective activity. This series of compounds can significantly improve zebrafish heart distortion and pericardial edema symptoms, improve zebrafish cardiac function, and significantly reduce zebrafish mortality in the adriamycin-induced myocardial injury zebrafish model. Mechanism studies have found that this series of compounds can protect the heart by restoring and reducing the MDA and GSH levels of zebrafish with myocardial injury, and downregulating the iron ion content in the zebrafish with myocardial injury and the mRNA level of the iron death marker gene PTGS2. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of the optimal dosing concentrations of compounds LSD, 1, 2a, 2c, 2k, 2l, and 2z in the zebrafish myocardial injury model. ####p<0.0001 compared with the control group, ****p<0.0001 compared with the model group.

[0028] Figure 2 Results of cardiac function evaluation of compounds LSD, 1, 2a, 2c, 2k, 2l, and 2z on fluorescently labeled zebrafish. ###p<0.001, ####p<0.0001 compared with the control group, **p<0.01, ****p<0.0001 compared with the model group. (A) Representative optical microscope images of zebrafish (scale bar = 500 μm) and fluorescent microscope images of fluorescent hearts (scale bar = 100 μm); (B) heart rate statistics; (C) shortening fraction statistics; (D) stroke volume statistics; (E) cardiac output statistics.

[0029] Figure 3The toxicity test results of compounds 2a and 2z on zebrafish embryo model. (A) Effects of different concentrations of compounds 2a and 2z on the survival rate of zebrafish embryos; (B) Bright field images of zebrafish after treatment with 100 μM 2a and 2z (scale bar = 500 μm).

[0030] Figure 4 The results of the effects of compounds 2a and 2z on doxorubicin-induced ferroptosis in zebrafish. ##p<0.01,###p<0.001,####p<0.0001 compared with the control group, **p<0.01,***p<0.001,****p<0.0001 compared with the model group. (A) PTGS2 mRNA levels in zebrafish of each group; (B) Iron content statistics in zebrafish of each group; (C) MDA levels in zebrafish of each group; (D) GSH levels in zebrafish of each group. DETAILED DESCRIPTION

[0031] The present invention is further explained below in conjunction with implementation and accompanying drawings, but implementation does not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the present invention are commercially available.

[0032] Example 1 Synthesis of Compounds 2a-z

[0033] As shown in synthetic route 1, steviol (1) is used as a starting material and reacts with a halogenated alkane compound in a potassium carbonate and acetone solution to generate compounds 2a-2z.

[0034] Reaction conditions and reagents in synthetic route 1: (a) halogenated compound, potassium carbonate, acetone, heating under reflux at 60°C.

[0035]

[0036] Wherein R in compound 2a-z is one of the following substituents:

[0037]

[0038] The specific steps are as follows:

[0039] (1) Synthesis of compounds 2a-z

[0040] Steviol (100 mg, 0.31 mmol), potassium carbonate (42.85 mg, 0.31 mmol), benzyl bromide (101.34 mg, 0.93 mmol) and acetone (3 mL) were added to a 10 mL round-bottom flask, heated to 60 ° C and refluxed with stirring for 2 h, saturated sodium chloride and ethyl acetate (volume ratio 1:1) were added to the reaction solution for extraction to obtain an organic layer, which was dried with anhydrous sodium sulfate at room temperature for 12 h and then concentrated. The concentrate was separated and purified by column chromatography to obtain one of the pure products 2a-z respectively;

[0041] Among them: the synthesis of 2a uses m-fluorobenzyl bromide, 2b uses 4-trifluoromethylbenzyl bromide, 2c uses 2,3-difluorobenzyl bromide, 2d uses 3,5-difluorobenzyl bromide, 2e uses o-fluorobenzyl bromide, 2f uses p-chlorobenzyl bromide, 2g uses m-chlorobenzyl bromide, 2h uses 2,4-difluorobenzyl bromide, 2i uses m-iodobenzyl bromide, 2j uses p-fluorobenzyl bromide, 2k uses 3-methylbenzyl bromide, 2l uses 3-nitrobenzyl bromide, 2m uses m-bromobenzyl bromide, 2n uses 3-phenylbenzyl bromide, 2 o uses 3-trifluoromethylbenzyl bromide, 2p uses 2,3,4,5,6-pentafluorobenzyl bromide, 2q uses 3,4-difluorobenzyl bromide, 2r uses 3,4-dimethoxybenzyl bromide, 2s uses 3-methoxybenzyl bromide, 2t uses 2,5-difluorobenzyl bromide, 2u uses 3-difluoromethoxybenzyl bromide, 2v uses o-chlorobenzyl bromide, 2w uses 3-cyanobenzyl bromide, 2x uses 3,4-dichlorobenzyl bromide, 2y uses 3-trifluoromethoxybenzyl bromide, and 2z uses 3-formylbenzyl bromide.

[0042] The structure, appearance, and NMR spectrum data of compounds 2a-z are shown below:

[0043] The structural formula of compound 2a is as follows:

[0044]

[0045] Compound 2a: white solid (174 mg, 87%), 1 H NMR(CDCl3,400MHz)δ7.40-7.02(4H,m,2,3,4,6-Ph),5.19-4.94(3H,overlap,CH2Ph,H- 17),4.81(1H,s,H-17),1.20(3H,s,H-18),0.78(3H,s,H-20),2.25–0.81(20H,m,CH,CH2 in ent-kaurene skeleton); 13C NMR (CDCl3, 100MHz) δ177.1,156.1,138.6,130.1,130.1,123.6,115.1,114.9,102.9,80.3,65.2,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 36 FO3,427.2648[M+H + ]; found,427.2648.

[0046] The structural formula of compound 2b is as follows:

[0047]

[0048] Compound 2b: white solid (176 mg, 79%), 1 H NMR(CDCl3,400MHz)δ7.70-7.60(2H,m,3,5-Ph),7.51-7.43(2H,m,2,6-Ph),5.26-5.01(2H,m,H-19), 4.97(1H,s,H-17),4.81(1H,s,H-17),1.21(3H,s,H-18),0.79(3H,s,H-20),2.28–0.80(20H,m,CH,CH2 in ent-kaureneskeleton); 13 C NMR(CDCl3,100MHz)δ177.1,156.1,140.1,128.2,125.6,125.6,125.5,125.5,103.0,80.2,65.1,57.0,53.7, 47.4,47.0,44.0,41.6,41.3,40.6,39.3,39.2,38.0,29.7,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 28 H 35 F3O3Na,476.2616[M+Na + ]; found,477.2653.

[0049] The structural formula of compound 2c is as follows:

[0050]

[0051] Compound 2c: white solid (164 mg, 79%), 1 H NMR(CDCl3,400MHz)δ7.22-7.00(3H,m,4,5,6-Ph),5.24-5.05(2H,m,CH2Ph),4.97(1H,s, H-17),4.81(1H,s,H-17),1.19(3H,s,H-18),0.77(3H,s,H-20),2.24–0.78(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.1,125.7,125.6,125.5,124.1,117.5,117.3,102.9,80.2,59.5,57.0,53. 7,47.4,46.9,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.8,20.4,19.0,15.3; HRMS(ESI,m / z)calcd for C 27 H 35 F2O3,445.2554[M+H + ]; found,445.2581.

[0052] The structural formula of compound 2d is as follows:

[0053]

[0054] Compound 2d: white solid (173 mg, 83%), 1 H NMR(CDCl3,400MHz)δ6.89-6.76(3H,overlap,2,4,6-Ph),5.15-4.96(3H,overlap,CH2Ph, H-17),4.81(1H,s,H-17),1.22(3H,s,H-18),0.79(3H,s,H-20),2.23–0.82(20H,m,CH,CH2 in ent-kaurene skeleton); 13C NMR (CDCl3, 100MHz) δ176.9,156.1,110.8,110.7,110.5,103.7,103.5,103.2,103.0,80.2,64.6,57.0,53. 7,47.4,47.0,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 35 F2O3,445.2554[M+H + ]; found,445.2581.

[0055] The structural formula of compound 2e is as follows:

[0056]

[0057] Compound 2e: white solid (160 mg, 80%), 1 H NMR(CDCl3,400MHz)δ7.39-7.29(2H,m,3,4-Ph),7.15-7.07(2H,m,5,6-Ph),5.20-5.07(2H,m,CH2Ph), 4.97(1H,s,H-17),4.80(1H,s,H-17),1.19(3H,s,H-18),0.76(3H,s,H-20),2.21–0.79(20H,m,CH,CH2 in ent-kaureneskeleton); 13 C NMR (CDCl3, 100MHz) δ177.1,156.2,131.0,130.2,130.2,124.1,115.6,115.3,102.9,80.3,60.1,57.1,53. 8,47.4,46.9,43.9,41.6,41.3,40.7,39.3,39.2,38.0,28.8,21.7,20.4,19.1,15.2; HRMS(ESI,m / z)calcd for C 27 H 36 FO3,427.2648[M+H + ]; found,427.2659.

[0058] The structural formula of compound 2f is as follows:

[0059]

[0060] Compound 2f: white solid (154 mg, 74%),1 H NMR(CDCl3,400MHz)δ7.37-7.27(4H,overlap,2,3,5,6-Ph),5.15-4.90(3H,overlap,CH2Ph ,H-17),4.81(1H,s,H-17),1.18(3H,s,H-18),0.77(3H,s,H-20),2.23–0.80(20H,m,CH,CH2 in ent-kaurene skeleton); 13 CNMR(CDCl3,100MHz)δ177.1,156.1,134.6,134.0,129.7,128.7,103.0,80.2,65.2,57.0,53.7,47.4,46. 9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,30.2,29.7,28.8,21.9,20.4,19.1,15.4; HRMS(ESI,m / z)calcd for C 27 H 36 ClO3,443.2353[M+H + ]; found,443.2381.

[0061] The structural formula of compound 2g is as follows:

[0062]

[0063] Compound 2g: white solid (168 mg, 81%), 1 H NMR(CDCl3,400MHz)δ7.36-7.23(4H,m,2,4,5,6-Ph),5.12-4.97(3H,overlap,CH2Ph,H- 17),4.81(1H,s,H-17),1.20(3H,s,H-18),0.77(3H,s,H-20),2.19–0.82(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.1,138.1,134.4,129.8,128.3,128.3,126.2,102.9,80.2,65.1,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H36 ClO3,443.2353[M+H + ]; found,443.2311.

[0064] The structural formula of compound 2h is as follows:

[0065]

[0066] Compound 2h: white solid (160 mg, 77%), 1 H NMR(CDCl3,400MHz)δ7.39-7.35(1H,m,6-Ph),6.89-6.84(2H,m,3,5-Ph),5.16-5.01(2H,m,CH2Ph),4 .97(1H,s,H-17),4.81(1H,s,H-17),1.17(3H,s,H-18),0.75(3H,s,H-20),2.20–0.79(20H,m,CH,CH2 in ent-kaureneskeleton); 13 C NMR (CDCl3, 100MHz) δ177.1,156.2,132.1,111.4,111.2,104.3,104.0,103.8,102.9,80.3,59.6,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.7,20.4,19.0,15.3; HRMS(ESI,m / z)calcd for C 27 H 35 F2O3,445.2554[M+H + ]; found,445.2587.

[0067] The structural formula of compound 2i is as follows:

[0068]

[0069] Compound 2i: white solid (195 mg, 78%), 1H NMR(CDCl3,400MHz)δ7.73(1H,s,2-Ph),7.66(1H,d,J=8.0Hz,4-Ph),7.33(1H,d,J=8.0Hz,6-Ph),7.10(1H,t,J=8.0Hz,5-Ph ),5.07-4.97(3H,overlap,CH2Ph,H-17),4.81(1H,s,H-17),1.20(3H,s,H-18),0.77(3H,s,H-20),2.22–0.79(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.1,156.2,138.4,137.2,137.1,130.2,127.4,102.9,94.2,80.2,65.0,57.0,53. 7,47.4,47.0,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 36 IO3,535.1709[M+H + ]; found,535.1767.

[0070] The structural formula of compound 2j is as follows:

[0071]

[0072] Compound 2j: white solid (170 mg, 85%), 1 H NMR(CDCl3,400MHz)δ7.36-7.27(2H,m,Ph-2,6)7.07-7.02(2H,m,Ph-3,5),5.13-4.96(3H,overlap ,CH2Ph,H-17),4.80(1H,s,H-17),1.18(3H,s,H-18),0.76(3H,s,H-20),2.20–0.79(20H,m,CH,CH2 in ent-kaureneskeleton); 13C NMR (CDCl3, 100MHz) δ177.1,163.8,161.3,156.1,131.9,130.3,115.5,115.3,102.9,80.2,65.3,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 36 FO3,427.2648[M+H + ]; found,427.2691.

[0073] The structural formula of compound 2k is as follows:

[0074]

[0075] Compound 2k: white solid (154 mg, 78%), 1 H NMR(CDCl3,400MHz)δ7.28-7.10(4H,m,2,4,5,6-Ph),5.09-4.93(3H,overlap,CH2Ph,H-17),4.81 (1H,s,H-17),2.36(3H,s,PhCH3),1.20(3H,s,H-18),0.77(3H,s,H-20),2.27–0.79(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR(CDCl3,100MHz)δ177.2,156.2,138.1,136.0,129.0,128.8,128.4,125.3,102.9,80.2,66.1,57.1,53.8, 47.5,46.9,43.9,41.6,41.4,40.7,39.3,39.2,38.1,28.9,21.9,21.4,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 28 H 38 O3Na,423.2899[M+Na + ]; found,423.2891.

[0076] The structural formula of compound 21 is as follows:

[0077]

[0078] Compound 21: white solid (125 mg, 59%),1 H NMR(CDCl3,400MHz)δ8.26(1H,s,2-Ph),8.22-8.17(1H,d,4-Ph),7.72-7.52(2H,m,5,6-Ph),5.28-5.08(2H,m,C H2Ph),4.97(1H,s,H-17),4.81(1H,s,H-17),1.22(3H,s,H-18),0.78(3H,s,H-20),2.28–0.78(20H,m,CH,CH2in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.0,148.4,138.3,133.9,129.6,123.1,122.9,103.0,80.2,64.6,57.0,53. 7,47.4,47.0,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,22.0,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 36 NO5,454.2593[M+H + ]; found,454.2618.

[0079] The structural formula of compound 2m is as follows:

[0080]

[0081] Compound 2m: white solid (171 mg, 75%), 1 H NMR(CDCl3,400MHz)δ7.53-7.23(4H,m,2,4,5,6-Ph),5.11-4.97(3H,m,CH2Ph,H-17) ,4.81(1H,s,H-17),1.20(3H,s,H-18),0.77(3H,s,H-20),2.21–0.79(20H,m,CH,CH2 in ent-kaurene skeleton); 13C NMR (CDCl3, 100MHz) δ177.2,156.2,138.1,136.0,129.0,128.8,128.4,125.3,102.9,80.2,66.1,57.1,53. 8,47.5,46.9,43.9,41.6,41.4,40.7,39.3,39.2,38.1,28.9,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 36 BrO3,487.1848[M+H + ]; found,487.1859.

[0082] The structural formula of compound 2n is as follows:

[0083]

[0084] Compound 2n: white solid (125 mg, 55%), 1 H NMR(CDCl3,400MHz)δ7.63-7.31(9H,m,2,4,5,6-Ph,2,3,4,5,6-Ph`),5.15(2H,q,J=12.0Hz,CH2Ph), 4.96(1H,s,H-17),4.80(1H,s,H-17),1.22(3H,s,H-18),0.79(3H,s,H-20),2.28–0.80(20H,m,CH,CH2 in ent-kaureneskeleton); 13 C NMR(CDCl3,100MHz)δ177.2,156.1,141.5,140.8,136.6,128.9,128.8,127.5,127.2,127.1,127.1,126.9,102.9,80.2,6 6.1,57.1,53.8,47.4,47.0,41.0,41.7,41.4,40.7,39.4,39.3,38.1,28.9,22.0,20.4,19.1,15.5; HRMS(ESI,m / z)calcd forC 33 H 41 O3,485.3055[M+H + ]; found,485.3114.

[0085] The structural formula of compound 2o is as follows:

[0086]

[0087] Compound 2o: white solid (123 mg, 55%), 1 H NMR(CDCl3,400MHz)δ7.70-7.41(4H,m,2,4,5,6-Ph),5.23-5.05(2H,m,CH2Ph),4.97(1H,s ,H-17),4.81(1H,s,H-17),1.21(3H,s,H-18),0.76(3H,s,H-20),2.26–0.77(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR(CDCl3,100MHz)δ177.1,156.1,137.1,131.4,129.1,124.9,124.9,124.8,103.0,80.2,65.1,57.0,53 .7,47.4,46.9,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,22.020.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 28 H 36 F3O3,477.2616[M+H + ]; found,477.2621.

[0088] The structural formula of compound 2p is as follows:

[0089]

[0090] Compound 2p: white solid (142 mg, 61%), 1 H NMR(CDCl3,400MHz)δ5.27-5.02(2H,m,CH2Ph),4.98(1H,s,H-17),4.81(1H,s,H-17),1.17(3H,s,H-18),0.80(3H,s,H-20),2.26–0.81(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR(CDCl3,100MHz)δ176.8,156.1,103.0,80.2,57.0,53.7,53.1,47.4,46.9,44.0,4 1.6,41.2,40.6,39.3,39.2,37.9,28.7,21.6,20.4,19.0,15.1; HRMS(ESI,m / z)calcd for C 27 H 31 F5O3Na,499.2271[M+Na+ ]; found,499.2296.

[0091] The structural formula of compound 2q is as follows:

[0092]

[0093] Compound 2q: white solid (148 mg, 71%), 1 H NMR(CDCl3,400MHz)δ7.18-7.12(3H,m,2,5,6-Ph)5.17-4.98(3H,m,CH2Ph,H-17),4.98(1H, s,H-17),4.81(1H,s,H-17),1.19(3H,s,H-18),0.77(3H,s,H-20),2.28–0.79(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.1,151.4,149.0,133.1,124.4,117.4,117.3,103.0,80.2,64.7,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 34 F2O3Na,445.2554[M+Na + ]; found,445.2587.

[0094] The structural formula of compound 2r is as follows:

[0095]

[0096] Compound 2r: white solid (182 mg, 77%), 1 H NMR(CDCl3,400MHz)δ6.97-6.81(3H,m,2,5,6-Ph)5.14-4.93(3H,m,CH2Ph,H-17),4.81(1H,s,H-17), 3.88(6H,s,3-Ph-OCH3,4-Ph-OCH3),1.19(3H,s,H-18),0.78(3H,s,H-20),2.25–0.79(20H,m,CH,CH2 in ent-kaureneskeleton); 13C NMR(CDCl3,100MHz)δ177.2,156.1,148.9,128.7,121.1,111.6,111.0,102.9,80.2,66.0,57.0,55.9,55.9,5 3.7,47.4,47.0,43.9,41.6,41.3,40.7,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 29 H 41 O5,469.2954[M+H + ]; found,469.2971.

[0097] The structural formula of compound 2s is as follows:

[0098]

[0099] Compound 2s: white solid (181 mg, 88%), 1 H NMR(CDCl3,400MHz)δ7.30-7.24(1H,m,2-Ph),7.00-6.71(3H,m,4,5,6-Ph),5.18-4.15(3H,m,CH2Ph,H-1 7),4.81(1H,s,H-17),3.81(3H,s,OCH3),1.21(3H,s,H-18),0.79(3H,s,H-20),2.26–0.80(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR(CDCl3,100MHz)δ177.2,159.7,156.1,137.7,129.6,120.3,113.6,113.5,102.9,80.2,65.9,57.1,55.2, 53.8,47.4,47.0,43.9,41.6,41.3,40.7,39.3,39.2,38.1,28.9,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 28 H 38 O4,439.2848[M+H + ]; found,439.2821.

[0100] The structural formula of compound 2t is as follows:

[0101]

[0102] Compound 2t: white solid (169 mg, 81%), 1 H NMR(CDCl3,400MHz)δ7.11-7.01(3H,m,3,4,6-Ph),5.17-5.03(2H,m,CH2Ph),4.97(1H,s, H-17),4.81(1H,s,H-17),1.20(3H,s,H-18),0.77(3H,s,H-20),2.25–0.81(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.1,124.9,117.0,116.8,116.7,116.4,116.3,102.9,80.2,59.5,57.0,53. 7,47.4,46.9,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.8,20.4,19.0,15.3; HRMS(ESI,m / z)calcd for C 27 H 34 F2O3Na,467.2374[M+Na + ]; found,467.2381.

[0103] The structural formula of compound 2u is as follows:

[0104]

[0105] Compound 2u: white solid (149 mg, 67%), 1 H NMR(CDCl3,400MHz)δ7.40-7.03(4H,m,2,4,5,6-Ph),6.73-6.32(1H,m,OCHF2),5.19-5.00(2H,m,CH2Ph ),4.97(1H,s,H-17),4.81(1H,s,H-17),1.21(3H,s,H-18),0.77(3H,s,H-20),2.25–0.78(20H,m,CH,CH2 in ent-kaureneskeleton); 13C NMR(CDCl3,100MHz)δ177.1,156.1,151.3,138.3,130.0,124.9,119.1,118.4,115.8,113.3,103.0,80.2,65.2 ,57.0,53.7,47.4,46.9,44.0,41.6,41.3,40.6,39.3,38.0,28.8,22.0,20.4,19.0,15.5; HRMS(ESI,m / z)calcd for C 28 H 37 F2O4,475.2660[M+H + ]; found,475.2681.

[0106] The structural formula of compound 2v is as follows:

[0107]

[0108] Compound 2v: white solid (180 mg, 87%), 1 H NMR(CDCl3,400MHz)δ7.64-7.45(4H,m,3,4,5,6-Ph),5.20-5.02(2H,m,CH2Ph),4.97(1H,s ,H-17),4.81(1H,s,H-17),1.20(3H,s,H-18),0.75(3H,s,H-20),2.25–0.78(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR(CDCl3,100MHz)δ177.0,156.0,137.7,132.5,131.8,131.6,129.5,118.5,112.8,103.0,80.2,64.7,57 .0,53.7,47.4,46.9,44.0,41.6,41.2,40.6,39.3,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 36 ClO3,443.2353[M+H + ]; found,443.2387.

[0109] The structural formula of compound 2w is as follows:

[0110]

[0111] Compound 2w: white solid (128 mg, 63%), 1H NMR(CDCl3,400MHz)δ7.52-7.21(4H,m,2,4,5,6-Ph),5.29-5.06(2H,m,CH2Ph),4.97(1H,s ,H-17),4.81(1H,s,H-17),1.21(3H,s,H-18),0.77(3H,s,H-20),2.25–0.78(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.1,156.1,134.0,133.7,130.4,129.5,129.5,126.8,102.9,80.2,63.5,57.0,53. 7,47.4,46.9,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.8,20.4,19.1,15.3; HRMS(ESI,m / z)calcd for C 28 H 36 NO3,434.2695[M+H + ]; found,434.2721.

[0112] The structural formula of compound 2x is as follows:

[0113]

[0114] Compound 2x: white solid (165 mg, 74%), 1 H NMR(CDCl3,400MHz)δ7.55-7.12(4H,m,2,4,5,6-Ph),5.12-4.92(3H,overlap,CH2Ph,H- 17),4.81(1H,s,H-17),1.19(3H,s,H-18),0.78(3H,s,H-20),2.25–0.78(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.0,136.4,132.7,132.2,130.6,130.2,127.5,103.0,80.2,64.5,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 27 H 35Cl2O3,477.1963[M+H + ]; found,477.1968.

[0115] The structural formula of compound 2y is as follows:

[0116]

[0117] Compound 2y: white solid (171 mg, 74%), 1 H NMR(CDCl3,400MHz)δ7.45-7.10(4H,m,2,4,5,6-Ph),5.20-5.00(2H,m,CH2Ph),4.97(1H,s ,H-17),4.81(1H,s,H-17),1.21(3H,s,H-18),0.77(3H,s,H-20),2.25–0.78(20H,m,CH,CH2 in ent-kaurene skeleton); 13 C NMR (CDCl3, 100MHz) δ177.0,156.1,149.4,138.4,130.0,126.3,120.3,120.4,103.0,80.2,65.0,57.0,53. 7,47.4,46.9,43.9,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.0,15.4; HRMS(ESI,m / z)calcd for C 28 H 36 F3O4,493.2565[M+H + ]; found,493.2597.

[0118] The structural formula of compound 2z is as follows:

[0119]

[0120] Compound 2z: white solid (141 mg, 69%), 1 H NMR(CDCl3,400MHz)δ10.03(1H,s,CHO),7.98-7.44(4H,m,2,4,5,6-Ph),5.29-5.02(2H,m,CH2Ph),4 .97(1H,s,H-17),4.81(1H,s,H-17),1.21(3H,s,H-18),0.76(3H,s,H-20),2.25–0.78(20H,m,CH,CH2 in ent-kaureneskeleton); 13C NMR(CDCl3,100MHz)δ192.0,177.1,156.1,137.4,136.7,134.1,129.6,129.3,129.2,103.0,80.2,65.2,57.0, 53.7,47.4,46.9,44.0,41.6,41.3,40.6,39.3,39.2,38.0,28.8,21.9,20.4,19.1,15.5; HRMS(ESI,m / z)calcd for C 28 H 37 O4,437.2692[M+H + ]; found,437.2708.

[0121] In order to demonstrate the advantages of the technical solution provided by the present application, an experimental example of the technical solution provided by the present application is given below.

[0122] Experimental Example 1 Study on the activity of steviol derivatives in zebrafish and exploration of the mechanism of compounds 2a and 2z

[0123] (1) Zebrafish culture and embryo collection: The genotypes of zebrafish were wild-type (Wild-type ABstrain) and cardiac fluorescent marker (Tg[cmlc2:GFP]), which were purchased from the National Zebrafish Resource Center Experimental Technology. Zebrafish were cultured and mated according to the National Zebrafish Resource Center Experimental Technology. The system was maintained under a 14-hour light / 10-hour dark cycle in an automatic circulation tank system and fed with live saltwater shrimp twice a day. The circulating water culture system of Haisheng Biological Experimental Equipment Company was used, with supporting filtration and impurity removal devices, chlorine removal and pure water devices, oxygenation devices, ultraviolet sterilization and disinfection devices, temperature control devices, etc. NaHCO3 and NaCl were added to the water circulation to maintain the conductivity of 500-550μS / cm, the pH range of 7.0±0.5, the water temperature range of 28±1℃, the dissolved oxygen of 6.0-8.0mg / L, and the NH3 concentration of less than 0.02mg / L. Zebrafish were placed in pairs in mating tanks at night and separated by walls of the same name. The walls were removed the next morning. Embryos were collected 90 minutes later, 150-200 per dish, placed in 0.0002% (w / v) methylene blue disinfectant solution, cultured in an incubator for 24 hours, and deformed or dead embryos were picked out using a stereo microscope. Washed three times with HolfBuffer and set aside.

[0124] (2) Drug activity screening: 24 hours after fertilization, zebrafish embryos were transferred to a plate containing 24 wells, 20 embryos were randomly assigned to each well, and 4 replicates were set up in each group for drug administration. The 24-well plate was placed in a constant temperature (28±0.5℃) incubator. At 96hpf, the survival rate of zebrafish was evaluated under an inverted fluorescence microscope. Both the atria and ventricles of zebrafish lost their contractility and were considered dead fish.

[0125] The results are shown in Table 1. 70 μM DOX can induce 50% death of zebrafish at 72 hpf. Compared with the doxorubicin (DOX) model group, the survival rate of zebrafish recovered to 68% after treatment with 40 μM levosimendan (LSD) in the positive control group. The survival rate of zebrafish recovered to 70% after treatment with 40 μM maternal steviol (1). Compounds 2a, 2c, 2k, 2l and 2z can effectively improve the survival rate of zebrafish.

[0126] Table 1 Effects of steviol derivatives on the survival rate of zebrafish

[0127]

[0128]

[0129] (3) Investigation of effective doses of compounds: Based on the results in Table 1, five compounds with excellent activity were found: 2a, 2c, 2k, 2l and 2z. Next, the transgenic zebrafish Tg (cmlc2:GFP) that specifically express green fluorescent protein in the heart was further used to evaluate the effects of these five compounds on the recovery of zebrafish cardiac function. First, the modeling concentration and optimal dosing concentration of the new model were explored. It was found that 70μM DOX at 48hpf would cause 50% of zebrafish death, the optimal dosing doses of 1 and LSD were both 60μM, and the optimal dosing doses of steviol derivatives 2a, 2c, 2k, 2l, and 2z were 2, 10, 5, 0.1, and 10μM ( Figure 1 ).

[0130] (4) Evaluation of zebrafish cardiac function: Fix the zebrafish with green fluorescent heart labeled at 72 hpf in methylcellulose fixative (4% concentration) and make it lie on its side to ensure that the eyes on both sides overlap and the body is on the same horizontal plane. Use an upright fluorescence microscope with a magnification of 50 times to observe and record the data. Immediately switch to the EGFP fluorescence channel and take and record the heart image under an upright fluorescence microscope with a magnification of 100 times. Calculate the number of heart beats in the video to get the heart rate of the zebrafish. Then, select adjacent contraction and relaxation frames from the video and export the corresponding images. Use the formula v = 4 / 3πab 2The ventricular volumes were calculated, and further the stroke volume, contraction fraction, and total stroke volume were calculated.

[0131] like Figure 2 As shown, after doxorubicin modeling, zebrafish showed distorted body shape, severe pericardial edema and slender heart. After treatment with levosimendan LSD, maternal steviol (1) and 2c, the heart morphology and pericardial edema recovered slightly, but not completely. After treatment with 2k, the zebrafish morphology and heart morphology recovered to be similar to the blank control group, but the pericardial edema was still very serious. 2a, 2l, and 2z restored the morphology, pericardial edema, and heart morphology of zebrafish to the same level as the blank control group. By statistically analyzing the heart function indicators of zebrafish, heart rate, shortening fraction, stroke volume, and cardiac output, it can be found that the cardioprotective activity of these five compounds in the zebrafish myocardial injury model is: 2z≈2a>2i>2k>2c>1>LSD, among which 2z almost recovered to be consistent with the blank control group.

[0132] (5) Preliminary drug toxicity evaluation in zebrafish: Wild-type zebrafish embryos fertilized after 24 hpf were transferred to a 96-well plate, with one embryo placed in each well. Each group contained 10 wells and was divided into a blank group and a drug test group. The morphological changes of zebrafish in the control blank group were observed every 24 hours, and the morphology of zebrafish in each group at 96 hpf was recorded. Figure 3 As shown, compounds 2a and 2z had no toxic side effects on zebrafish embryos at concentrations ranging from 5 μM to 100 μM, and the body shape of the zebrafish was the same as that of the control group.

[0133] (6) Determination of PTGS2 mRNA level in zebrafish: RNA was extracted from 72 hpf zebrafish, and the RNA concentration was measured using Nanodrop2000S. The mRNA was reverse transcribed into cDNA using a reverse transcription kit and placed on ice. The mRNA was amplified and the level was detected on a PCR series analyzer using the SYBR dye method. The primer sequences are as follows: PTGS2-Forward, 5`-TGGATCTTTCCTGGGTGAAGG-3`; PTGS2-Reverse, 5`-GAAGCTCAGGGGTAGGTGCAG-3`. The results are shown in Figure 2. Figure 4 As shown in A, 2a and 2z can dose-dependently downregulate the mRNA level of ferroptosis marker gene PTGS2.

[0134] (7) Detection of iron content in zebrafish: The embryo processing steps (2) were repeated, and the zebrafish at 72 hpf were killed. The iron content in each group of zebrafish was detected according to the instructions of the iron content detection kit (Nanjing Jiancheng). The results are shown in Figure 4 As shown in B, 2a and 2z can dose-dependently downregulate the iron content in zebrafish.

[0135] (8) Detection of GSH levels in zebrafish: Repeat the embryo processing steps (2), kill the zebrafish at 72 hpf, and perform the test according to the instructions of the kit. Add an appropriate amount of PBS buffer to the tissue and perform ultrasonic homogenization on ice. Take 0.1 mL of the suspension after the crushing, add 0.1 mL of reagent 1 and mix well. Centrifuge at 3500 rpm for 10 minutes, and take the supernatant for testing. Prepare the detection working solution according to the instructions, add the sample and the detection working solution to a 96-well plate, mix well, let it stand at room temperature for 5 minutes, and use an enzyme reader to measure the absorbance value of each well at 405 nm. The results are as follows. Figure 4 As shown in C, 2a and 2z could dose-dependently upregulate the antioxidant GSH.

[0136] (9) Detection of MDA levels in zebrafish: The embryo processing steps were repeated as in (2). The zebrafish at 72 hpf were killed and tested according to the instructions of the kit. An appropriate amount of lysis buffer was added to the tissue, and the tissue was lysed on ice for 4 hours and then disrupted with an ultrasonic disruptor. An appropriate amount of TBA was weighed and prepared with TBA diluent to prepare a TBA working solution with a concentration of 0.68%. The MDA detection working solution was prepared according to the instructions. 100 μL of lysis buffer and 200 μL of MDA detection working solution were added to a centrifuge tube. Heat in a metal bath at 100°C for 15 minutes. Cool to room temperature, centrifuge at 1000g for 10 minutes at room temperature, take 200 μL of supernatant and add it to a 96-well plate, and measure the absorbance at 532 nm using an enzyme reader. The results are as follows. Figure 4 As shown in D, 2a and 2z could dose-dependently down-regulate the level of MDA, a lipid peroxide.

Claims

1. A steviol derivative, characterized in that: It has the general structural formula 1: Formula 1; Wherein: R is one of m-fluorobenzyl, 4-trifluoromethylbenzyl, 2,3-difluorobenzyl, 3,5-difluorobenzyl, o-fluorobenzyl, p-chlorobenzyl, m-chlorobenzyl, 2,4-difluorobenzyl, m-iodobenzyl, p-fluorobenzyl, 3-methylbenzyl, 3-nitrobenzyl, m-bromobenzyl, 3-phenylbenzyl, 3-trifluoromethylbenzyl, 2,3,4,5,6-pentafluorobenzyl, 3,4-difluorobenzyl, 3,4-dimethoxybenzyl, 3-methoxybenzyl, 2,5-difluorobenzyl, 3-difluoromethoxybenzyl, o-chlorobenzyl, 3-cyanobenzyl, 3,4-dichlorobenzyl, 3-trifluoromethoxybenzyl, and 3-aldehydebenzyl.

2. The method for preparing the steviol derivative according to claim 1, characterized in that: The steps are as follows: (1) Synthesis of compounds 2a-z: 0.47 mmol of compound 1, 0.7 mmol of potassium carbonate and 0.7 mmol of halogenated compound were dissolved in 3 mmol of acetone, and the mixture was stirred under reflux at 60°C for 5 h; a mixture of ethyl acetate and saturated brine was added for extraction, and the organic layer was collected, dried over anhydrous sodium sulfate, concentrated, and separated and purified by column chromatography to obtain compounds 2a-z; Its synthetic route is as follows: ； Wherein R in compound 1 and compound 2a-z is one of the following substituents: 。 3. Use of the steviol derivative according to claim 1 in the preparation of a cardioprotective drug.

4. A cardioprotective drug comprising the steviol derivative according to claim 1.

5. A cardioprotective drug according to claim 4, further comprising a pharmaceutically acceptable excipient.

6. The cardioprotective drug according to claim 4, wherein the excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH adjuster.

7. A cardioprotective drug according to claim 4, wherein the drug is in the form of injection, tablet, oral solution, granule or capsule.

Images